The role of direct heparan sulfate interactions and Ca2+ complexation in Hedgehog gradient formation in vivo

发育生物学中的一个主要问题是细胞如何协调它们与邻居的行为。作为发育过程中细胞交流的中心模型，Hedgehog(HH)形态原从它们的来源扩散到果蝇的远距离反应细胞和组织中直接生长和图案形成。未受损的HH转运和梯度形成需要细胞外表达线性和高负电荷的糖链，称为硫酸乙酰肝素(HS)。然而，HS结合并固定了大多数其他细胞外可溶蛋白，提出了与HS的相互作用如何促进HH传播而不是减缓HH传播的问题。在上一个资助期，我们解决了这个悖论，表明HH与细胞表面HS的直接静电相互作用启动和引导HH的运输，其方式类似于静电限制的DNA结合蛋白在细胞核中的运动。DNA结合蛋白最好沿着双螺旋的轴线移动，两个DNA结合位点允许糖-磷酸骨架之间的直接转移，以提高搜索速度。我们结合了硅胶、体外和体内的先进技术，证明了HHS也确实通过两个密集排列的正电荷在梯度场中的许多糖-硫酸盐链之间直接切换，并且一个位置上的正电荷减少严重地损害了HH的体外切换和体内向远距离靶点的运输。因此，我们揭示了直接在HS链之间切换的能力代表了以前未知的时间编码的形态原运动的决定因素，对其他HS结合蛋白具有重要的意义。在这项提议中，我们的目标是利用我们建立的体外和体内实验方案，扰乱HH无序N-尾部中带电氨基酸残基的组成和位置。我们还将研究HS在发育中的组织中的动态表达和硫酸盐化，这决定了形态生物质的可达“微空间”。我们预计，在其周围组织上增加HS硫酸盐化的HS微空间会积累HH，而表达HS的较低硫酸盐化的邻近微空间不会进入。我们将使用针对特定HS结构的单链可变片段(ScFv)抗体来可视化发育中的翼和眼盘微腔中HS的动态表达和硫化变化。我们的第三个目标是继续描述缺乏与钙离子协调的HH氨基酸的苍蝇品系。这些额度是在上一个资助期使用我们的既定方案产生的。它们的表型表明，HH-Ca~(2+)配位对果蝇的发育不是必需的，但对于干细胞增殖和果蝇配子发生是特殊需要的。因此，在下一个资助期，我们的目标是进一步描述HH生物学的这一重要但鲜为人知的方面。

A major question in developmental biology is how cells coordinate their behaviors with that of their neighbors. As a central model for cellular communication during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in distant responding cells and tissues in Drosophila. Unimpaired Hh transport and gradient formation require extracellular expression of linear and highly negatively charged sugar chains called heparan sulfate (HS). However, HS binds and immobilizes most other extracellular soluble proteins, raising the question of how interactions with HS contribute to Hh spread instead of slowing it down. In the last funding period, we resolved this paradox by showing that direct electrostatic Hh interactions with cell-surface HS initiate and guide Hh transport in a similar way to electrostatically constrained DNA-binding protein movement in the nucleus. DNA-binding proteins preferably move along the axis of the double helix, and two DNA binding sites allow for direct transfer between sugar-phosphate backbones to increase search speed. Using a combination of in silico, advanced in vitro and in vivo techniques, we showed that Hhs do also switch directly between the many sugar-sulfate chains in the gradient field via two sites of densely arranged positive charge, and that positive charge reduction in one site severely impairs Hh switching in vitro and its transport to distant targets in vivo. Thus, we revealed that the ability to directly switch between HS chains represents a previously unknown determinant of temporally encoded morphogen movement with important implications for other HS-binding proteins. In this proposal, we aim to perturb the composition and position of charged amino acid residues in the disordered N-tail of Hh, using our established in vitro and in vivo protocols. We will also investigate dynamic HS expression and sulfation in developing tissues that determines accessible “microspaces” for morphogens. We expect that HS microspaces of increased HS sulfation over their surrounding tissue accumulate Hh, whereas neighboring microspaces expressing HS with lower sulfation will not be entered. We will visualize dynamic HS expression and sulfation changes in developing wing and eye disc microspaces by using single chain variable fragment (scFv) antibodies directed against defined HS structures. Our third aim is the continued characterization of fly lines made deficient in Ca2+-coordinating Hh amino acids. These lines were generated in the last funding period using our established protocol. Their phenotypes revealed that Hh Ca2+-coordination is non-essential for fly development, but is specifically required for stem cell proliferation and fly gametogenesis. In the next funding period, we therefore aim to further characterize this important yet poorly understood aspect of Hh biology.